Vertebrate cells contain at least 2 isoforms of nonmuscle myosin II heavy chains (MHCs), MHC-A and MHC-B and expression of these two isoforms is regulated in a tissue-dependent manner. Previously, we have reported that chicken and human MHC-B can contain cassettes of 10 and 21 amino acids in the loop 1 and loop 2 region, respectively (Takahashi et al., J. Biol. Chem. 267, 17864, 1992). The expression of MHC-B containing either or both of these two inserts is associated with neuronal cell differentiation and brain development (Itoh and Adelstein, Ibid 270, 14533, 1995). In this study, we attempt to investigate the cellular function of these specific isoforms in the cell. First, we construct an expression vector containing cDNA which encodes each inserted isoform of heavy meromyosin (HMM) (amino acids 1-1045). Transfection of this cDNA results in transient high expression of these isoforms in two mammalian cell lines, HeLa and COS7. Second, we made stable transfected cells which continuously expressed each inserted isoform of HMM and investigated these cells. Expressed HMM is soluble in the high salt and ATP containing extraction buffer and shows a similar cellular localization, especially to the peripheral region of the cell, as does endogenous MHC-B. We also screened for target protein(s) using the yeast two hybrid system. A number of cDNA clones which can interact with expressed HMM containing either of these two inserts were identified. One of these is FUS protein, which is fused to the transcription factor CHOP following translocation (12:16). This abnormal fusion protein is associated with malignant liposarcoma (Rabbitts et al., Nature Genetics 4, 175, 1993). We synthesized a full- length cDNA encoding FUS using RT-PCR and HeLa cell mRNA. We then expressed the FUS protein fused to a FLAG peptide at the C-terminus using the mammalian expression system and the Baculovirus system and will study its interaction with MHC-B containing the 21 amino acid insert.